Proteins exported via the primary export pathway of the gram negative bacterium Escherichia coli are synthesized as precursors with amino- terminal signal peptides that are thought to have major roles in the export process, including entry into the pathway and translocation across the cytoplasmic membrane. To date, many, perhaps all, of the protein components of the export pathway have been identified. However, the precise role of these components and how they interact with one another, membrane lipids, and the signal peptide and mature moiety of precursor proteins to facilitate the export process have yet to be determined. For the past few years, our laboratory has been intensively investigating the synthesis and secretion of the periplasmic maltose-binding protein (MBP) of Escherichia coli using a combination of genetic and biochemical approaches. Our long term goal is to understand, at the molecular level, the mechanism by which the MBP is exported from its site of synthesis in the cytoplasm to the periplasm. The following are our specific aims for the proposed five year project period: (1) We will continue our analysis of the MBP signal peptide with particular emphasis on the cleavage site and in understanding why certain mutant signal peptides promote unusually rapid MBP export. (2) Studies on the cleavage site of preMBP have provided two new genetic selections that may yield mutants with alterations in SPase I or other known or unknown components of the export machinery. (3) The recognition and translocation of export-defective MBP species in E. coli cells harboring various pr1 suppressor alleles will be investigated further. (4) We will determine if SecB specifically recognizes the mature moiety of precursor MBP, or if such an interaction is not sequence specific and/or is partly determined by the signal peptide or other factors. (5) Experiments are planned to distinguish between the chaperone and targeting functions of SecB, including attempts to isolate mutations in secA or secB that specifically inhibit the targeting activity. (6) In an effort to understand why some proteins are exported in a SecB-independent manner, we will expand our studies addressing the localization of the periplasmic ribose-binding protein.